Sensitive Detection of the 4977-bp Deletion in Human Mitochondrial DNA of Young Individuals

Specific deletions in mitochondrial DNA (mtDNA) are present at high levels in skeletal muscle and other tissues (7). It has been suggested that an accumulation of these somatic deletions could be caused by oxidative damage that increases with age (2). In recent years, more than 20 different types of deletions have been identified using polymerase chain reaction (PCR) techniques (16). The most frequently observed deletion is 4977-bp long and occurs at a presumed deletion “hot spot” involving two 13-base direct repeats beginning at positions 8469 and 13 447 in the mitochondrial genome sequence (4). This deletion shows a marked correlation to age (6,11) and has also been found in human brain tissue of patients with Parkinson’s disease (8), in heartmuscle tissue of patients with cardiomyopathy (12), in skeletal muscle of patients with Kearns-Sayre syndrome (17) and in several other degenerative diseases (13). The undeleted mtDNA and the basal level of the 4977-bp deletion can be detected under specific PCR conditions (4,9). In the past few years, the amplification products were detected on agarose gels followed by ethidium bromide staining (9). Unfortunately, this technique does not detect very small quantities of DNA, and the signal is not permanent (15). Highly sensitive detection of nucleic acids in the picogram range has been achieved by the specific chemical reduction of silver ions (5). Our former study showed that the smaller the deletion-specific fragments, the better the detection threshold (unpublished observations), because smaller fragments have a higher amplification efficiency (14). For this reason, we chose new primer sequences that were located nearer to the deletion breakpoints, so that smaller fragments could be amplified from the deleted mitochondrial genome (Figure 1). The use of these new primers and the detection on ultrathin polyacrylamide gels (PAG) led to an improved detection threshold and to the detection of the 4977-bp deletion in subjects younger than 20 years without any mitochondrial diseases, which has not yet been described by others. Autopsy tissues were obtained from individuals representing a wide range of ages. Total DNA was isolated from 100 mg of skeletal muscle (musculus iliopsoas) using the Super QUIKGENE SQG1 Kit (Immucor, Rödermark, Germany) following the manufacturer’s instructions. The amount of DNA was quantified by the Human DNA Quantitation System (Life Technologies, Gaithersburg, MD, USA). This slot-blot method is specific for human nuclear DNA and is very sensitive, with a detection limit of 20 pg DNA. In contrast to photometric quantitation, it makes possible the determination of the concentration of highly degraded DNA. Unfortunately this method does not detect mtDNA. Currently, to our knowledge, there is no mtDNA-specific slot blot for a routine analysis available. Assuming that nearly 1% of total extracted DNA is mtDNA, we used the nuclear-specific slot blot to get an ap-